PROJECT SUMMARY Following exposure to a virus, the immune system activates and takes rapid measures to protect the body from a spreading infection. Our understanding of the signaling cascade that leads to immuno-activation in response to viral infections is crucial for designing efficient therapeutic drugs that regulate this process. Cell surface and cytosolic receptors recognize pathogen-specific molecules such as viral nucleic acids that activate the interferon regulator factors 3 (IRF3), which is a master regulator of immune activation. Once activated, IRF3 promotes the expression of interferon ? (IFN?), which is essential for the antiviral response. Despite IRF3's active role in antiviral response, dysregulation in IRF3 expression has been implicated in multiple autoimmune diseases. Strict regulation of IFN? is critical to minimize uncontrolled inflammation damage to infected hosts while clearing the infection. Together with ATF2/c-Jun, p65/p50, and IRF7, IRF3 form a higher order molecular complex called the IFN? enhanceosome to strictly regulate IFN? expression. While the principles of the signaling pathway that activates IRF3 and induces IFN? expression are established, structural insights into the activation of IRF3 and IFN? enhanceosome assembly remain scarce. In this proposal, I seek to characterize the structural mechanism of IRF3 activation by using X-ray crystallography. Then, I will use cryo-electron microscopy to characterize the structural features promoting IRF3 interactions with p65 at the enhanceosome. These studies will provide insights into how the other IRFs get activated and how other enhanceosome complexes assemble. Most importantly, structural-based analyses of IRF3 activation and assembly with p65 will provide a basis for designing small molecule modulators that can directly target the activation and suppression of IRF3 in response to viral infections and autoimmune diseases, respectively. !